Throughout this application, various patents and papers are referenced. The disclosures thereof in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.
This invention relates to the field of blood group determination, and particularly to the simultaneous determination of forward and reverse blood group testing.
Blood group serology requires the determination of blood cell compatibility between a blood donor and a patient recipient before a transfusion or organ transplant involving the patient. Blood cell compatibility is determined by the non-occurrence of an immunological reaction between antibodies contained in the blood serum of a patient and antigens present on blood cells from the donor.
Many different blood group antigens are found on the surface of the red blood cells of every individual. These antigens, the products of inherited genes, exist in combinations that are likely to be unique between all individuals except identical twins. Blood grouping is generally the process of testing red cells to determine which antigens are present and which are absent, normally utilizing antibodies to the antigen tested for. Additionally, when a person does not have a particular red cell antigen on his or her red blood cells, his or her serum may contain an antibody to that antigen. Whether or not the antibody is present in the serum depends on whether the person's immune system has been previously challenged by, and responded to, that specific antigen or something very similar to it. For example, a person whose red blood cells are Type A, i.e., having “A” antigens on the red cells, will have anti-B antibodies in his or her serum. Thus, if such a person is given type B blood, an immunological reaction will occur with possible serious clinical consequences.
As an additional consideration, it should be noted that the human body is constantly exposed to antigens in pollens, food, bacteria and viruses. Some of these “natural” antigens are apparently so similar to human blood group antigens that they stimulate almost every susceptible person to produce antibodies. Thus, certain antibodies are expected in the serum of anyone whose red cells lack the reciprocal antigen. This is especially true of the ABO system. Accordingly, a second confirmatory test is often performed on the patient/donor sera. The test for expected antibodies of the ABO blood group system in sera is called “reverse” blood grouping.
Antibodies of the ABO blood grouping system are generally immunoglobulin M (IgM). These antibodies have ten antigen binding sites per molecule. The IgM antibody is large enough to span the distance between red blood cells, so that when they are centrifuged, the cells will be bound together in a lattice “cell-antibody-cell-antibody” and will remain clumped together in agglutinates. For example, if anti-A is added to blood group A or blood group AB cells and the mixture is centrifuged, the cells will remain in clumps when resuspended. With the same antibody, group 0 and group B cells will resuspend as individual cells. Agglutination caused by one antibody, such as an IgM antibody, is called direct agglutination.
In transfusion medicine, the most frequently performed test, for the reasons given above, is determination of the ABO blood group. The current state of the art is separately testing for A, B, and sometimes A+B together, antigens on the red cells (forward type); and confirmation (cross-check) testing for anti-A and anti-B antibodies in serum or plasma (reverse type). Thus a minimum of 4 separate tests, but as many as 7 separate tests (A, B, A+B antigens on the sample red cells; anti-A and anti-B in the sample serum/plasma using A1, A2, B, O reagent red cells) are routinely employed. The results from each of these typing exercises (forward and reverse types) have to agree. Thus, in the U.S. alone approximately 104 million tests are performed annually to determine the blood groups in blood centers.
Since the early 1900's, the general approach known as the “Landsteiner” method, (Landsteiner, Science 73:405 (1931)) together with the work of Ashby, (J. Exp. Med. 29:267 (1919)) and Coombs (Brit. J. Exp. Pathol. 26:255 (1945)), has been to add a patient's red blood cells to a standard laboratory test tube containing a blood group antibody (such as Anti-A or Anti-B), mix to allow antibody/antigen binding reactions to take place, and then to centrifuge. If the antigen tested for is present, the antibody/antigen binding will have taken place resulting in agglutination of the patient's red blood cells. The test tube is manually shaken to dislodge the centrifuged button of “clumped” cells at the bottom. A subjective determination is then made as to whether or not the dislodged cells are “clumped”, and to what extent.
During the mid-1900's, attempts were made to simplify this technique to minimize the subjective nature of the test and to reduce mistakes. It was recognized that a somewhat permanent record of the results of compatibility testing could be had by employing wettable, either non-absorbent or in some cases absorbent, test slides or test cards having the requisite immunochemical reagents on at least a portion of their surfaces. In this regard, U.S. Pat. Nos. 2,770,572, 2,850,430, 3,074,853, 3,272,319, 3,424,558, 3,502,437 and 3,666,421 European Patent Application #0 104 881-A2 depict select examples of such test cards and related apparati. The advantages of blood grouping in microplates include easier manipulation of large numbers of samples, objective measurement of agglutination reactions using instruments and computer interfacing for compilation and management of results. A number of expensive and dedicated systems with computer-controlled robotics and high throughput spectrophotometric readers have been introduced for blood bank automation (Chung, et al., Transfusion 33:384 (1992)).
Commercial blood typing kits have been introduced with improved detection of red cell antigen and antibody reactions in special microtubes filled with reagents in gel form. (Lapierre, et al., Transfusion 30:109 (1990)). Utility of solid phase techniques to overcome the inherent problems in the use of hemagglutination as an end point for blood grouping was reviewed by Scott (Transfusion Med. Rev. 5:60 (1991)). Recently Growe et al. (Transfusion Med. Rev. 10:44 (1996)) reported the implementation and use of automated grouping of RBC antigen and serum antibody screening procedures applicable not only to blood centers but also for hospital transfusion laboratories. At least seven different wells with specific typing reagents have to be used in these methods for determining the blood group of a sample. All of the procedures used for large scale blood grouping essentially employed the agglutination based methods and also for the determination of only one antigen or antibody type in a single tube/well. Separately identifiable reactions using fluorescent labeled reagents have also been reported for blood typing applications in the U.S. Pat. Nos. 4,550,017 and 4,748,129.
Current procedures utilize agglutination of red cells as an endpoint. As discussed hereinabove, this is accomplished in test tubes, on slide surfaces, in microplates and in column agglutination tests. The latter 2 methods may be performed manually or by automated instrumentation. All methods require separation of serum (or plasma) from cells to perform both forward and reverse type.
It is therefore of interest to develop a method that performs both forward and reverse type in a single test, while preferably avoiding the need to separate the blood sample prior to testing. Such a method would make it possible for a blood bank technologist to simultaneously determine blood group antigens on red cells as well as antibodies of clinical significance in serum. Such a method would considerably decrease the number of individual tests performed i.e., provide a reduction of about 50 to 100 million tests per year in the number of tests performed in blood centers resulting in significant savings of time and cost. We have developed techniques to allow discrimination of the sample red blood cells (RBCs) from reagent RBCs so that their agglutinates are distinct. Additionally, the novel methods disclosed herein use labeled typing antibody reagent to distinguish their reaction from preformed antibody in the sample. The inventive tests are done without the need to separate the blood sample and thus can be done on whole blood (WB). The tests disclosed herein can be used on automated instruments, where further advantage is gained by not having to separate cells from serum. Further, the simultaneous detection of forward and reverse test reduces the number of tests required to type and confirm the type (forward & reverse test).
In the fluorescent labeling embodiment of the invention, in the forward test, the monoclonal antibody is labeled. For the reverse test the reagent red cells are labeled. Further advantage rests in the ability to distinguish (visually or via automated reader) mixed red cell populations. For instance, reverse test can be performed in 1 test instead of 2, resulting in a reduction of number of tests performed. A further application is the antibody screen, where a pool of 2 cells could be used together instead of 2 separate tests, resulting in a reduction of number of tests performed.
The ability to color cells for a visual system has applications to existing blood group test platforms using tubes, microplates, slides and Column Agglutination Technology (CAT), and test platforms such as the Vitros™ 250, 750 or 950 (Ortho-Clinical Diagnostics, Inc., Rochester, N.Y.) slide method.
Vyas et al. In U.S. Pat. No. 5,776,711 disclose a “simultaneous” ABO and RH(D) blood typing or antibody screening method. However, while similarly attempting to reduce the number of tests to perform an ABO blood group, the instant invention simultaneous performs the samples' red cell ABO status as well as presence of antibodies to A and/or B antigens. In addition, the instant invention utilized whole blood rather than separated serum and red cell components. The instant use of labeled reagent red cells also differs from the Vyas et al. use of synthetic beads.
Simultaneous analysis of ABO blood group can be carried out by labeling reagent red blood cells with appropriate fluorochromes and selecting appropriate monoclonal antibodies with fluorochrome labels. The cells and agglutinates can be measured by laser scanning cytometry on a microscope slide or equivalent. The cells on the slide are illuminated by a scanning laser light source. Typically, laser light sources used include blue argon ion lasers and/or red helium-neon lasers. Fluorescence and light scatter can be determined by the use of a Laser Scanning Cytometer (Compucyte, Cambridge, Mass.).
The use of laser scanning cytometry in the simultaneous determination of forward and reverse test is as follows. When a cell or group of cells (agglutinate) is scanned by the laser light beam, the illuminating light is scattered by the cell or group of cells and the intensity of scatter relates to cell (or agglutinate) size and shape. For example, individual red cells scatter less light than small agglutinates which in turn, scatter less light than large agglutinates.
Likewise, when a cell or group of cells (agglutinate) is scanned by the laser light beam, the illuminating light can induce fluorescence from fluorochrome(s) that have been associated with the cell. If a fluorochrome is relatively uniformly associated per cell with reagent red cells, the fluorescence intensity relates to agglutinate size. For example, individual red cells would fluoresce less than small agglutinates which in turn, would fluoresce less than large agglutinates.
Use of the combination of scattered light and fluorescence is more reliable than either parameter alone in discriminating different classes of agglutinates.
In addition to these two parameters, monoclonal antibodies that have been conjugated with a fluorochrome also may be used to label the cells (and agglutinates) of interest. The fluorescence emitted by the cells when excited by the illuminating laser beam yields additional information about the binding of these monoclonal antibodies to cells or agglutinates for distinguishing subpopulations of cells or agglutinates.
Five parameter (forward scatter, side scatter, and three fluorescence channels) dot plot analysis for simultaneous ABO determination is presented in FIGS. 1A–D.
It may be appreciated by those skilled in the art that flow cytometry or fluorescence microscopy could also be used to perform simultaneous analysis of ABO blood group.
In flow cytometry, the cells and agglutinates to be measured are introduced into the center of a fast moving fluid stream and forced to flow single file out of a small diameter orifice at uniform speeds. The particles are hydrodynamically focused to the center of the stream by a surrounding layer of sheath fluid. The cells within the stream pass a measurement station where they are illuminated by a light source and measurements are made at rates of 2.5×102 to 106 cells per minute. Laser light sources are used in the measurement of cells; typical laser light sources used include argon ion lasers (UV, blue and green light), krypton lasers (yellow and red light), helium-cadmium lasers (UV and blue light), and helium-neon lasers (red light). Fluorescence and light scatter can be determined by the use of a flow cytometer, for example, the CytoronAbsolute™ Flow Cytometer (Ortho-Clinical Diagnostics, Inc., Raritan, N.J.).
In fluorescence microscopy, the cells and agglutinates to be measured are read on a microscope slide or equivalent. The cells are typically illuminated by a white light source or a substantially monochromatic light source. Here, too, laser light sources may be used as the source of the monochromatic light. The presence of agglutinates may be assessed with the white light, and the associated fluorescence assessed with the monochromatic light and appropriate filters. Visual or automated reading may be used for one or both of these readings.
A visual detection technology can also be employed in the forward and reverse blood typing contemplated herein. Such a method, using a column agglutination test (CAT), may employ a BioVue™ cassette (Ortho-Clinical Diagnostics, Inc., Rochester, N.Y.). Such cassette contains columns to which has been added a microparticle matrix. Such matrix can be overlayed with suitable blood typing antibodies dispersed in buffer, forming the initial reaction zone.
CAT can employ automated reader systems to interpret the agglutination result. One such reader is present in the Ortho AutoVue™ (Ortho-Clinical Diagnostics, Inc., Rochester, N.Y.) a fully automated system to perform the CAT. The autoreader is a computerized imaging system consisting of a CCD (charged coupled device) monochrome video camera, and image processing board, and an IBM-compatible PC. The reader first acquires an image of the reaction that is digitised and processed by the image-processing software to extract the reaction features, which are then used by the reaction classification program. These features are used to separate reactions into negative and positive classes, and for translation into one of seven conventional reaction classes or grades. Discriminate analysis, a linear statistical pattern recognition tool, is used to distinguish between negative and weak reactions.
Yet another reader employed in CAT is the BioVue™ Reader 2 (Ortho-Clinical Diagnostics, Inc., Rochester, N.Y.). This Reader has an automated loader for twelve BioVue™ cassettes and has a halogen lamp source and image analysis features permitting cell identification based on RBC wavelength thereby interpreting the agglutination result. Image acquisition is performed by a CCD camera and a digitising board.